LSD1/PRMT6-targeting gene therapy to attenuate androgen receptor toxic gain-of-function ameliorates spinobulbar muscular atrophy phenotypes in flies and mice

Spinobulbar muscular atrophy (SBMA) is caused by CAG expansions in the androgen receptor gene. Androgen binding to polyQ-expanded androgen receptor triggers SBMA through a combination of toxic gain-of-function and loss-of-function mechanisms. Leveraging cell lines, mice, and patient-derived specimens, we show that androgen receptor co-regulators lysine-specific demethylase 1 (LSD1) and protein arginine methyltransferase 6 (PRMT6) are overexpressed in an androgen-dependent manner specifically in the skeletal muscle of SBMA patients and mice. LSD1 and PRMT6 cooperatively and synergistically transactivate androgen receptor, and their effect is enhanced by expanded polyQ. Pharmacological and genetic silencing of LSD1 and PRMT6 attenuates polyQ-expanded androgen receptor transactivation in SBMA cells and suppresses toxicity in SBMA flies, and a preclinical approach based on miRNA-mediated silencing of LSD1 and PRMT6 attenuates disease manifestations in SBMA mice. These observations suggest that targeting overexpressed co-regulators can attenuate androgen receptor toxic gain-of-function without exacerbating loss-of-function, highlighting a potential therapeutic strategy for patients with SBMA.

and PRMT6 are overexpressed selectively in SBMA skeletal muscle. a) Western blot analysis of LSD1 and PRMT6 in the indicated tissues of 12-week-old WT and AR100Q mice (n = 4 mice/genotype). LSD1 and PRMT6 were detected with specific antibodies, and CNX was used as loading control. Molecular weight is indicated on the right. b) (Left) RT-PCR analysis of LSD1 and PRMT6 transcript levels normalized to ACTIN in the liver of SBMA patients and healthy controls (n = 3 control and 3 patientderived biopsies).
(Right) RT-PCR analysis of LSD1 and PRMT6 transcript levels normalized to HPRT1 in patient-derived induced pluripotent stem cells (iPSCs) differentiated to motor neurons (n = 2 control and 3 SBMA patient-derived cell lines). c) Chromatin-immunoprecipitation assays of AR occupancy at a nonandrogenresponsive chromatin locus (negative control) and at the known AR-regulated gene, Fkbp5 (positive control) in C2C12 myoblasts expressing AR24Q or AR100Q and treated with vehicle or DHT (10 nM, 12 h). Shown is one experiment representative of three technical replicates. Graphs, mean ± sem, two-tailed student's t test. Source data are provided as a Source Data file. interaction in HEK293T cells (n = 3 biological replicates). AR was detected with anti-Flag antibody and LSD1 with a specific antibody. Molecular weight is indicated on the right. Source data are provided as a Source Data file. Supplementary Fig. 3. Overlapping subcellular localization of endogenous LSD1 with normal and polyQ-expanded AR. a-c) Immunofluorescence analysis of the subcellular localization of normal and polyQexpanded AR and LSD1 in MN1 cells treated with vehicle or DHT (10 nM, 16 h) (a), patient-derived iPSCs differentiated to motor neurons (b), and a spinal cord sample from a deceased SBMA patient (c). iPSCs also were stained for PRMT6 and motor neuron marker HB9. AR, PRMT6, LSD1, and HB9 were detected with specific antibodies, and nuclei were stained with DAPI. Shown are images representative of at least three independent experiments, except for panel c, which is the only spinal cord autopsy sample available. Scale bar = 20 µm (a) and 10 µm (b, c) d) Negative controls for proximity ligation assay (PLA) in MN1 cells expressing AR100Q and treated with DHT (10 nM, 16 h). Only the antibody against LSD1 (left), AR (middle), or PRMT6 (right) was added to the reaction. Scale bar = 17 μm, n = 3 biological replicates.
Supplementary Fig. 4. LSD1 is a co-activator of normal and polyQ-expanded AR. a) Transcriptional assays in HEK293T cells expressing AR12Q or AR55Q driven by the elongation factor 1α promoter alone or together with LSD1 and treated with vehicle or DHT (10 nM, 16 h; n = 4 biological replicates). b) Transcriptional assays in HEK293T cells expressing AR24Q or AR65Q alone or together with the indicated LSD1 isoforms and treated with vehicle or DHT (10 nM, 16 h; n = 4 biological replicates). c) Transcriptional assays in MN1 cells expressing AR24Q alone or together with the indicated LSD1 isoforms and treated with vehicle or DHT (10 nM, 16 h; n = 3 biological replicates). d) Western blot of LSD1 expression in HEK293T cells stably expressing Cas9 and the indicated guides targeting LSD1. Shown is one experiment representative of n = 7 biological replicates. LSD1 was detected with a specific antibody, and β-Tub was used as loading control. Graphs, mean ± sem, two-way ANOVA followed by Tukey HSD test. Source data are provided as a Source Data file.

Supplementary Fig. 5
Supplementary Fig. 5. LSD1 requires the AF-2 surface of AR and its catalytic activity to transactivate normal AR. a) Transcriptional assay in HEK293T cells expressing AR12Q or AR12Q-E897K alone (mock) or together with LSD1. Cells were treated with DHT (10 nM, 16 h; n = 3 biological replicates). b) Transcriptional assay in HEK293T cells expressing AR24Q alone (mock) or with either LSD1 or LSD1-LXXAA. Cells were treated with DHT (10 nM, 16 h; n = 6 mock and LSD1 overexpression, and n = 5 LSD1-LXXAA overexpression biological replicates). c) Transcriptional assay in HEK293T cells expressing AR24Q alone (mock) or with LSD1 or the catalytic inactive mutant LSD1-K685A. Cells were treated with DHT (10 nM, 16 h; n = 3 biological replicates). Graphs, mean ± sem, two-way ANOVA (a), one-way ANOVA (b, c) followed by Tukey HSD test. Source data are provided as a Source Data file. (Right) Western blot of LSD1 and PRMT6 in HEK293T cells transduced with lentiviral vectors to silence LSD1 and PRMT6 by CRISPR technology. Shown is one experiment representative of 3 (right) and 2 (left) biological replicates. Quantification is shown at the bottom. LSD1 and PRMT6 were detected with specific antibodies, and β-Tub was used as loading control.
b) Transcriptional assay in HEK293T cells expressing AR65Q or AR65Q-S215A,S792A alone (mock) or with LSD1 and/or PRMT6. Cells were treated with DHT (10 nM, 16 h; n = 3 biological replicates). c) Transcriptional assay in HEK293T cells with and without silencing guides targeting LSD1 and expressing AR24Q or AR65Q alone (mock) or with PRMT6. Cells were treated with DHT (10 nM, 16 h; n = 6 biological replicates). Graphs, mean ± sem, two-way ANOVA followed by Tukey HSD test. Source data are provided as a Source Data file. Supplementary Fig. 7. Efficacy of amiR target silencing in vivo. Western blots and corresponding quantification in the indicated tissues of 13-week-old AR100Q mice treated with and without amiR-Lsd1/Prmt6. Skeletal muscle LSD1 expression: n = 6 mice/treatment; PRMT6 expression: n = 3 mice/treatment; spinal cord, liver, adipose tissue and lungs: n = 4 mice/treatment; heart: n = 3 mice/treatment. LSD1, PRMT6, and GFP were detected with specific antibodies, and CNX and actin were used as loading controls. Graphs, mean ± sem, two-tailed student's t test. Source data are provided as a Source Data file. Supplementary Fig. 8 Supplementary Fig. 8. Effect of amiR treatment on gene expression. RT-PCR analysis of LSD1, AR, and PRMT6 target genes in AR100Q mice treated with vehicle or amiR-Lsd1/Prmt6 (n = 3 mice/genotype). Graphs, mean ± sem, two-tailed student's t test. Source data are provided as a Source Data file.